Determination of lipid signaling pathways and the production of some intracellular signaling molecules has been an focus of some research work in the field of signal transduction research. For example, the determination of the regulation of different phospholipase activity and the formation of intracellular second messengers and other lipid bioactive molecules from different sources is an important component in the studies on the biological effects and their mechanisms of growth factors and other molecules. Here, a method was introduced for selectively labeling different phospholipids, which has been widely used in the study of lipid signaling pathways. Experimental evidence was presented to demonstrate how to determine the different signaling pathway and the generation as well as the alteration of relevant signaling molecules. The application of this method is flexible and has a high reproducibility. It can be used to pinpoint some signaling pathways and the formation of different signaling molecules. A brief introduction about lipid signaling and its significance in the area of signal transduction was also given.